Ustilago hordei is a basidiomycetes which causes covered smut of barley. Asexual growth occurs in the form of non-pathogenic, haploid, budding yeast-like sporidia. Fusion of sporidia, which is controlled by a single mating-type locus with two alleles, results in a pathogenic, mycelial dikaryon. We have shown that mating involves communication between cells of the opposite mating type, resulting in the formation of conjugation tubes. A very exciting finding is that small molecular weight compounds, or pheromones, act as signals. We have isolated numerous mutants from both mating types which are incapable of mating. Some of these mutants are unable to produce active pheromone and others are incapable of making conjugation tubes. The long range goal of this research effort is to biochemically characterize purified pheromones and clone genes involved in this pheromone mediated cell-cell interaction in order to better understand the basic mechanisms involved in the conversion of a non- pathogen to a pathogen. The MBRS student involved in this project will continue work started under the previous grant. The formation of stable U. hordei diploids by complementation of auxotrophic mutants was demonstrated. This opens the door to using parasexual genetics to analyze this host-pathogen interaction. Previous genetic analyses have shown this interaction to be governed by a gene-for-gene relationship. The MBRS student working on this project will isolate ade mutants of U. hordei which display a red color phenotype. Since this mutation is recessive, diploids will be hyaline. However, rehaploidization will yield red sectors in colonies of diploid cells. These red haploids will be isolated and analyzed to determine segregation of auxotrophic markers, mating-type and a virulence genes. This will be the first step toward cloning genes directly involved in virulence of this pathogenic fungus and its eukaryotic host.